Cardiomyocyte apoptosis is now accepted to be a major contributor to cell death induced by ischemia/[unreadable] reperfusion (I/R) as well as to the development of dilated cardiomyopathy and heart failure. In the heart, as[unreadable] in isolated cardiomyocytes, the serine threonine kinase Akt protects against I/R injury and apoptosis induced[unreadable] by various cell stresses. Akt is activated through growth factor stimulation of PI3 kinase, an enzyme that has[unreadable] been well studied in the heart. The other enzymes that regulate Akt phosphorylation have not been explored[unreadable] in cardiomyocytes although these are likely drug targets for cardioprotection. These include the immediate[unreadable] upstream kinase PDK-1, and phosphatases responsible for Akt dephosphorylation amongst which is the[unreadable] recently discovered phosphatase, PHLPP. In Aim # 1 we address the regulation and role of PHLPP and the[unreadable] requirement for PDK-1 in Akt phosphorylation and activity in cardiomyocytes, using PHLPP and PDK-1[unreadable] knockout mice. We further examine how loss of these enzymes affects ischemic reperfusion damage in vivo[unreadable] and the development of apoptosis, mitochondria! disruption and autophagy in vitro. A great many studies of[unreadable] Akt have used activated membrane targeted forms of the enzyme. It has also become increasingly clear,[unreadable] however, that Akt has actions at multiple sites including the plasma membrane, cytosol, nucleus and[unreadable] mitochondria. The cellular location and dynamic control of Akt by its regulatory enzymes can lead to a range[unreadable] of spatiotemporal signals which will in turn dictate the nature of the downstream signaling events. Thus[unreadable] whether Akt activation regulates cytosolic enzymes, nuclear gene expression or mitochondrial integrity may[unreadable] depend upon its activation kinetics and location. Accordingly in Aim # 2 we propose to examine the temporal[unreadable] and spatial aspects of Akt regulation using a FRET based Akt activity reporter (BKAR) and other BKARS[unreadable] targeted to specific cellular locations. The possibility that mTOR acts not only as a downstream target of Akt[unreadable] but as a feedback regulator of Akt dephosphorylation will be examined in Aim # 3, testing the hypothesis that[unreadable] rapamycin affects Akt dephosphorylation through PHLPP or regulates kinase mediated Akt phosphorylation.[unreadable] The possibility that rapamycin affects the cells decisions to undergo autophagy through regulation of Akt will[unreadable] also be examined. We recently demonstrated that Akt associates with mitochondria in cardiomyocytes, and[unreadable] in particular with components of the mitochondrial permeability transition (PT) pore including hexokinase[unreadable] (HKII). In Aim # 4 the regulation of the HKII expression, its association with mitochondria, and its[unreadable] phosphorylation by Akt are examined. The role of HKII in mediating the cardioprotective effects of Akt on the[unreadable] mitochondrial permeability transition pore is tested. Elucidation of novel enzymatic pathways regulating Akt[unreadable] phosphorylation, and information on the compartmentalized effects of this enzyme, will allow design of[unreadable] therapeutic interventions that can selectively target components of this pleiotropic signaling pathway.